Trypsin
PurMa Biologics Trypsin Products
Trypsin is an enzyme critical in the digestive process, primarily involved in protein digestion. Produced in the pancreas and secreted into the small intestine, it exists initially as an inactive precursor, trypsinogen. Upon entering the small intestine, trypsinogen is activated into trypsin by another enzyme, enterokinase, found in the duodenum. It then breaks down long chains of amino acids, the building blocks of proteins, into smaller peptides and amino acids, facilitating their absorption and utilization by the body. As a serine protease from the PA clan superfamily, it’s function and activity are essential in maintaining normal digestive processes and overall protein metabolism.
Trypsin with EDTA
Trypsin Without EDTA
What is the protocol for enzymatic dissociation of monolayer cultures?
There are a few important points in dissociation of adhesive cells. Following the below mentioned instructions, the dissociation and making new plate for maintenance, transfection or other downstream application can easily be done while maintaining cellular integrity.
Before starting there is a few points need to be considered:
- For each particular commercial or primary cell line, please follow or guideline but the final and the most optimized conditions as well as concentrations used should be determined empirically.
- 24 hours after sub-culturing, the viability and well-being of the cells should be evaluated. That will save you time and prevent starting an experiment with an faulty plate. Cell viability should be more than 90%.
- The presence of at least 1-2 mm EDTA in the solution is crucial. PurMa Biologics produces various concentrations, with and without EDTA. That is because for some cell lines as well as some experiments EDTA interferes with the results.
- Make sure using the trypsin which has been prepared with water free of Mg+2 and Ca+2 as these ions compromise the functionality of this enzyme.
- The best temperature for keeping a bottle before the first use is -80°C. After thawing the bottle, prepare 10 ml aliquots using the 15ml falcon tubes and keep in -20°C and avoid freezing and thawing. The most important reason is that the potency of trypsin deteriorates after freezing and thawing which might not be realized by lab personnel’s but affect the reproducibility of result. The main reason for that is that with every use of trypsin the cell wall is injuring the cell wall, and the recovery time will be affected by potency of trypsin. So, using trypsin with different potencies can affect the recovery times.
Procedure
- Warm all reagents to 37°C prior to use.
- Spin down the cells and remove the growth medium from the cells.
- Rinse the palate containing cell monolayers with 5-7 ml Mg+2 and Ca+2 free PBS. Shake the flask (or dish), incubate the flask for 2-3 minutes at room temperature or 37°C CO2 incubator. Gently, tap flask or dish against the edge of the bench or any firm object. Check the flask under an inverted microscope. If cells are not yet detached and singular, allow to sit at room temperature for another 2 to 3 minutes and continue taping flask as described. Keep doing this until the cells are completely detached and seem dissociated from each other.
- Spin down the cells and remove the buffer from cells.
- Place 5 ml growth media and pipet well.
- Count the cells and pipet the appropriate amount in each flask or dish containing the appropriate amount of medium of choice containing FBS.
